Membrane lipid coated nanoparticles and method of use

ABSTRACT

Disclosed is a nanoparticle comprising an inner core comprising a virus; and an outer surface comprising a cellular membrane derived from a cell, and process of making thereof. The virus is an oncolytic virus and cellular membrane is derived from for example red blood cells.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/606,754 filed on Oct. 20, 2019, which is a 371 of InternationalPatent Application No. PCT/US18/28752, filed Apr. 21, 2018, which claimspriority to U.S. provisional application Ser. No. 62/488,685, filed Apr.21, 2017, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Oncolytic viruses (OVs) are viruses that preferentially infect and killcancer cells. The viruses grow and cause lysis (oncolysis) of cancercells or trigger other mechanisms to disturb thecancer-immunosuppressive microenvironment and trigger the body's immuneresponse to clear cancer cells. Recent successful clinical data and drugapprovals have increased public attention on oncolytic virotherapy. Theuse of oncolytic virotherapy can be combined with other drugs, immunecheck point inhibitors, and T-cell therapy to improve outcomes forcancer patients.

Routes of delivery of OVs include intratumoral (i.t.) injection,intravenous (i.v.) delivery, and intra-peritoneal delivery, whereintratumoral injection is applied mostly.

SUMMARY OF THE INVENTION

In accordance with the present invention, the present invention providesa process of making a particular nanoparticle comprising combining aninner core comprising a virus, or the like, and an outer surfacecomprising a cellular membrane derived from a cell in a non-salt watersolution; applying sonication to said solution to form a nanoparticlecomprising said inner core coated with said outer surface.

In one aspect, provided herein are nanoparticles comprising an innercore comprising a virus; and an outer surface comprising a cellularmembrane derived from a cell.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows the exemplary results of the virus detection assay ofdifferent sample batches prepared by applying various sonication timesof 4 minutes, 6 minutes, 8 minutes, respectively vs. bare virus.

FIG. 2 shows the exemplary results of the virus detection assay ofdifferent sample batches prepared by combining RBC membrane with a coreof a virus in various solutions in the nanoparticle preparation process.There are four sample batches (combining RBC membrane with the virusstock solution in saline, sucrose, dextrose, and lysine-dextrosesolution, respectively) and one control batch (bare virus).

FIG. 3 shows the TEM images of a bare virus and a coated virus.

FIG. 4 shows the sizes of bare virus and coated virus measured bydynamic light scattering.

FIG. 5 shows an exemplary virus detection assay result indicating thevirus was coated resulted a very low absorbance at 450 nm vs. one of thebare virus showing much higher absorbance.

FIG. 6 provides a follow up virus detection assay after the coated virusnanoparticles went through a de-coating process resulted in a bare virusin comparison with the coated virus.

DETAILED DESCRIPTION OF THE INVENTION

Currently, most oncolytic virus therapies are limited to local regionalinjection (such as intratumoral, i.t.), and whether a single injectionis enough to achieve therapeutic effect is still under investigation.Also, some OVs have been shown to have an acute, transient toxicityprofile, and thus injecting the virus may induce a competitive immuneresponse against the virus rather than against the tumor cells. Also,there is a problem for intravenous delivery of OVs where OVs may becleared rapidly from the bloodstream, thus requiring frequent orhigh-dose administrations, leading to increased therapy costs andpotential safety issues. Therefore, it is needed to provide acomposition comprising OVs that can be administered and remain incirculation for extended periods of time for ultimate delivery oftherapeutic agents to targeted cells.

It is known in the art that to achieve stealth moiety on nanoparticle,the adoption of polyethylene glycol (PEG) is employed. However, ananti-PEG immunological response may be triggered and thus such approachis problematic. Alternative approaches such as utilizing zwitterionicmaterials (e.g., poly(carboxybetaine) and poly(sulfobetaine)) have beenproposed.

Recently, a top-down biomimetic approach to provide functionalizednanoparticles by coating with natural erythrocyte membranes, includingboth membrane lipids and associated membrane proteins was realized,providing long-circulating cargo delivery. See C-M. J. Hu et al.,“Erythrocyte membrane-camouflaged polymeric nanoparticles as abiomimetic delivery platform,” Proc. Natl. Acad. Sci. USA 2011, Jul. 5;108(27): 10980-10985. Particularly, the membrane lipids derived from ablood cell (e.g., red blood cell (RBC), white blood cell (WBC), orplatelet) are of particular interests to coat various of materials butnot the oncolytic viruses, or the like.

Various attempts to prepare nanoparticles with RBC coated oncolyticviruses were tried but not successful following the known methods. It isthe first time that an oncolytic virus, or the like has been coated withRBC membrane. In accordance with the practice of the invention, it issurprisingly found that a certain condition must be employed to prepareinvention nanoparticles disclosed herein.

The term “oncolytic virus” referred herein includes non-limited examplesof herpesvirus; vaccinia virus; reovirus; adenovirus; measles virus,parvovirus, or combinations thereof.

It is known in the art that viruses can be used as vectors for deliveryof suicide genes, encoding enzymes that can metabolize a separatelyadministered non-toxic pro-drug into a potent cytotoxin, which candiffuse to and kill neighboring cells. Thus, the therapeutic agentdisclosed here also includes such vectors, suicide genes, or encodingenzymes.

In accordance with the present invention, it is found surprising aprocess to make a nanoparticle comprising combining an inner corecomprising a virus (a therapeutic agent), and an outer surfacecomprising a cellular membrane derived from a cell in a non-salt watersolution; applying sonication to said solution to form a nanoparticlecomprising said inner core coated with said outer surface. Contrary tothe known methods that a salt solution such as saline and PBS solutionis needed in the cellular membrane coating of the inner core process, anon-salt water solution (e.g., a sugar containing solution), or the like(such as a component with similar property of a sugar in watersolution), is required for coating a virus, such as an oncolytic virus.For the first time a nanoparticle comprising an inner core comprising avirus; and an outer surface comprising a cellular membrane derived froma cell is prepared in accordance with the practice of this invention. Itis surprising found that a 5% to 15%, a 7% to 14%, a 9% to 11% of anon-salt water solution (such as a sugar containing solution) used inthe process of preparing nanoparticles comprising a core of a virus, orthe like, is needed. In some embodiments, a non-salt water solution(such as a sugar containing solution) of 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, or 15% is needed in the process of making nanoparticlescomprising a core of a virus, or the like.

In some embodiments provide a nanoparticle comprising an inner corecomprising a virus; and an outer surface comprising a cellular membranederived from a cell. In certain embodiments, said virus is an oncolyticvirus. In certain embodiments, said oncolytic virus is herpesvirus;vaccinia virus; reovirus; adenovirus; measles virus, parvovirus, orcombinations thereof. In certain embodiments, said virus is adenovirus.

In some embodiments, said cell is a blood cell, an adipocyte, a stemcell, an endothelial cell, an exosome, a secretory vesicle or a synapticvesicle. In certain embodiments, said blood cell is red blood cell,white blood cell, or platelet. In certain embodiments, said blood cellis red blood cell.

In some embodiments provide a process of making a nanoparticlecomprising combining an inner core comprising a virus, and an outersurface comprising a cellular membrane derived from a cell in a non-saltwater solution; applying sonication to said solution to form ananoparticle comprising said inner core coated with said outer surface.In certain embodiments, said non-salt water solution comprises sugar, orthe like (such as a component with similar property of a sugar in watersolution). In certain embodiments, said non-salt water solution is asugar solution. In certain embodiments, said sugar solution is sucrose,or dextrose containing solution. In some embodiments, said virus is anoncolytic virus. In certain embodiments, said oncolytic virus isherpesvirus; vaccinia virus; reovirus; adenovirus; measles virus,parvovirus, or combinations thereof. In certain embodiments, saidoncolytic virus is adenovirus. In some embodiments, said cell is a bloodcell, an adipocyte, a stem cell, an endothelial cell, an exosome, asecretory vesicle or a synaptic vesicle. In certain embodiments, saidblood cell is red blood cell, white blood cell, or platelet. In certainembodiments, said blood cell is red blood cell.

The invention provides a cellular membrane derived nanoparticle fordelivering of a therapeutic agent, such as an oncolytic virus. The agentis essentially camouflaged by coating the therapeutic agent such asoncolytic virus with cellular membrane lipids.

In some embodiments, a therapeutic agent such as oncolytic virus iscoated with lipids compatible with circulation within the bloodstream ofa subject (e.g., a patient). This allows delivery of the agent to tumorvasculature via the EPR (enhanced permeability and retention) effect,where certain molecules tend to accumulate in tumor tissues more than innormal tissues. The EPR effect is usually employed to describenanoparticle and liposome delivery to cancer tissue. One of manyexamples is the work regarding thermal ablation with gold nanoparticles.Thus, in some embodiments, the nanoparticles disclosed herein wouldaccumulate near a tumor's vasculature, and the therapeutic agents (e.g.,a OV) will come into contact with cancer cells. The OV then infects thecell, resulting in cell lysis, death, or removal.

In some embodiments, provided herein are nanoparticles comprising aninner core comprising an oncolytic virus, and the like; and an outersurface comprising a cellular membrane derived from a cell such as ablood cell (e.g., red blood cell (RBC or erythrocyte), white blood cell,or platelet), an adipocyte, a stem cell, an endothelial cell, or thelike. In certain embodiments, the cellular membrane is derived from ablood cell such as red blood cell.

In some embodiments provide the method to prepare the nanoparticlesdisclosed herein. For example, blood cells can be purified from wholeblood or from processed red blood cells obtained from a blood supplier.RBCs are particularly useful as a membrane source because of theirabundance in human blood and the ease of collecting blood fromindividual donors. RBCs can be provided in the form of erythrocyteghosts (or RBC ghosts), where the internal proteins have been depleted,leaving the membrane components essentially intact. The use of RBCghosts also reduces the presence of RBC cellular proteins that mayinterfere during the coating process. In some embodiments, the RBC is atype O-negative blood cell (i.e., an “universal donor”). Such membranesource can be used in the preparation of the nanoparticles to deliverthe therapeutic agent (e.g., an OV) to a greater number of subjects. Incertain embodiments, one can use cell membranes derived from the samepatient for personalized batches of nanoparticles.

Compared to other platforms, for example a PEG coating nanoparticle, theRBC membrane derived nanoparticle has many surface markers such as CD47,CD59 (MAC-inhibitory protein, avoiding complement cell lysis), CD55(DAF), CD35 (CR1) and other members in immunoglobulin superfamily toprotect itself from body clearance. In some embodiments, the cellmembrane used for coating herein can further incorporate non-lipidcomponents, such as cell surface markers, MHC molecules, andglycoproteins. In other embodiments, the cell membrane can furtherincorporate hydrophilic components, such as PEG.

The term “cellular membrane” as used herein refers to a biologicalmembrane enclosing or separating structure acting as a selectivebarrier, within or around a cell or an emergent viral particle. Thecellular membrane is selectively permeable to ions and organic moleculesand controls the movement of substances in and out of cells. Thecellular membrane comprises a phospholipid uni- or bilayer, andoptionally associated proteins and carbohydrates. As used herein, thecellular membrane refers to a membrane obtained from a naturallyoccurring biological membrane of a cell or cellular organelles, or onederived therefrom.

As used herein, the term “naturally occurring” refers to one existing innature.

As used herein, the term “derived therefrom” refers to any subsequentmodification of the natural membrane, such as isolating the cellularmembrane, creating portions or fragments of the membrane, removingand/or adding certain components, such as lipid, protein orcarbohydrates, from or into the membrane taken from a cell or a cellularorganelle. A membrane can be derived from a naturally occurring membraneby any suitable methods. For example, a membrane can be prepared orisolated from a cell and the prepared or isolated membrane can becombined with other substances or materials to form a derived membrane.In another example, a cell can be recombinantly engineered to produce“non-natural” or “natural” substances that are incorporated into itsmembrane in vivo, and the cellular or viral membrane can be prepared orisolated from the cell to form a derived membrane.

A cellular membrane can be prepared by known methods. For example, cellscan be broken using a microfluidizer (MF), or a hypotonic solution, suchas water, followed by ultrafiltration or diafiltration with saline orPBS. In some cases, a solution with higher ionic strength has beenuseful for removing blood from pork liver, so a PBS or NaCl solution canhelp remove intracellular mass through the filtration process. Ananticoagulant, such as EDTA, can also be used, for example duringdiafiltration to remove impurities. In some embodiments, a TangentialFlow Filtration (TFF) device or centrifugation can be used to purify themembranes. The purified membrane components can be tested for thepresence of cellular proteins, such as by a BCA protein test.Preferably, most non-membrane components are removed prior to coating.

Despite the much efforts, it was found that following the known methods,such as the procedures in US2013/033 7066, a virus such as an oncolyticvirus or a CRISPR, a DNA sequence from viruses, cannot be coated orencapsulated by a cell membrane (e.g., a RBC membrane). It issurprisingly found that the cell membranes (e.g., RBC ghosts) coat thevirus, or the like, only in certain conditions. The process to preparethe nanoparticles disclosed herein requires (1) sonication of themixture of RBC ghosts and OVs in (2) non-salt solution such as a sucrosesolution.

In accordance with the practice of the invention, viruses suitable forcoating include oncolytic viruses as well as other viruses that caninfect a cancer cell. Viruses can have enveloped or noneveloped forms.In some embodiments, viral vectors are preferred because they can infecta cancer cell and replicate (replication competency).

Certain Pharmaceutical and Medical Terminology

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case may be determinedusing techniques, such as a dose escalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The term “pharmaceutical composition” refers to a mixture of ananoparticle (i.e., nanoparticle described herein) with other chemicalcomponents, such as, disintegrators, binders, lubricants, carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients. The pharmaceutical composition facilitatesadministration of the compound to an organism. Multiple techniques ofadministering a compound exist in the art including, but not limited to:intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary andtopical administration.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one embodiment, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

All of the various embodiments or options described herein can becombined in any and all variations. The following Examples serve only toillustrate the invention and are not to be construed in any way to limitthe invention.

EXAMPLES Example 1. Exemplary Preparation of Cell Membrane Preparation

The cell membrane preparation (e.g., a RBC ghost preparation) is knownthe art. Here a non-limited example of preparing a cell membrane, a RBCcell membrane, was followed to provide an exemplary cell membrane forinvention nanoparticles preparation.

Equipment and Materials Requirement:

Equipment/Accessories Millipore Labscale TFF System Millipore Pellicon 2mini TFF filter (PXDVPPC50) 0.5 mM EDTA DPBS (10X), no calcium, nomagnesium Packed RBC, ACD-A anticoagulant

Procedure:

-   -   1. Place the packed red blood cell bags in −80° C. fridge and        freeze overnight. Thaw packed red blood cell in 4° C. fridge in        the next morning.    -   2. Pour 360 mL of 0.5 mM EDTA into the media bottle, store in        fridge overnight.    -   3. Add 40 mL blood from pRBC to media bottle, shack to mix well.        Gentle mix in the fridge for 30 min.    -   4. Place 400 mL WFI water into TFF (Millipore Labscale TFF        System) process reservoir and close the reservoir. Start the TFF        system, turn the flow rate knob to level 4. Recirculate the        system for 5 min and push all the water through the permeate        line to pre-wet the filter (Millipore Pellicon 2 mini TFF        filter, PXDVPPC50). Pause the system.    -   5. Pour the mixture from step 3 into the TFF process reservoir.    -   6. Concentrate the mixture to 100 mL.    -   7. Diafiltration with 400 mL PBS. Slowly pour in the PBS into        the reservoir to keep the solution in the reservoir 100±50 mL.    -   8. Diafiltration with 200 mL 0.5 mM EDTA. Slowly pour in the        EDTA solution into the reservoir to keep the solution in the        reservoir 100±50 mL.    -   9. Final concentration to 40 mL. Monitor the pressure, lower the        pump speed to keep pressure<40 psi.    -   10. Pump out the lipid into a 50 mL conical tube, store in        −80° C. freezer for further analysis to confirm preparation of        cell membrane and/or processes.

Example 2: Failure Attempt to Prepare Nanoparticles Comprising a VirusCore Following Known Methods with Different Sonication Time Study

This procedure is describing the lab scale process for 1 mL coated virus(10{circumflex over ( )}10 VP/mL) using probe sonication. The excipientcell membrane (e.g., erythrocyte membrane) used in this process isequivalent to 0.1 mL of packed red blood cells as raw material. Thefollowing is a detailed description of attempting preparation ofnanoparticles comprising a virus core following the known method.

Because the first few attempts to prepare nanoparticles comprising acore of a virus following the known methods such as one in USPublication No. 2013/0337066 were not successful, it was decided toexplore the energy condition by applying different sonication time (4minutes, 6 minutes, and 8 minutes), hoping to prepare nanoparticlecomprising a core of an exemplary virus (i.e., human type 5 adenovirus).

Equipment and materials: the following is a brief description of theequipment and material used for synthesizing 1 mL coated virus:

Probe Sonicator: Qsonica XL2000

List of Raw Materials:

Name Description Notes Adenovirus Human type 5 adenovirus From vectorbiolabs Solution used Saline As described in the in the mixture knownmethod H₂O Sterile water for injection RBC Made from RBC Membrane 1.5mg/mL based membrane Derivation Process as in on BCA Example 1

Procedure

-   -   1. Add 800 uL of saline in a 1.5 mL microcentrifuge tube.    -   2. Add 200 uL of RBC membrane (1.5 mg/mL) into the tube, mix        well. This will make process mixture with 0.3 mg/mL membrane.    -   3. Turn on the power of probe sonicator, wipe the probe with 70%        ethanol and move only the probe into the biohood.    -   4. Adjust the power of the sonicator to level 2 (output˜3 W)    -   5. Prepare an ice box with cold water to keep the process        temperature at <4° C.    -   6. Add 10 uL adenovirus stock solution (Vector biolabs, Lot        #20170922) into the process mixture.    -   7. Put the tube into the ice box. Dip the probe into the tube.        Don't let the probe touch the bottom.    -   8. Manually sonicate the mixture with 1 second sonication and 1        second interval between each sonication. Maintain this step for        4, 6, and 8 min to produce three sample batches.    -   9. For the bare virus, follow steps 1-8, but change the solution        in 2. to molecular grade water.    -   10. Follow vendor's procedure (Virusys Corporation, AK290-2) to        obtain the sandwich ELISA data for each batch.

The coated virus nanoparticles were to be confirmed by the virusdetection assay. The assay is a double antibody (sandwich) ELISA thatutilizes a monoclonal anti-adenovirus antibody to capture the antigenfrom the sample. Following sample incubation, a biotinylated detectionantibody is added and this step is followed by HRP-Streptavidin. Thepresence of hexon antigen is visualized by the addition of an HRPsubstrate followed by the addition of a stop reagent. The development ofcolor within the well is indicative of the presence of adenovirus hexonantigen in the sample. For quantitative purposes, Virusys offers aseparate calibration kit (AK291, Adenovirus Antigen Calibration Kit)which can be used in conjunction with this product to generate astandard curve for quantitative measurement.

The results show that applying 4 minutes of sonication time seems toprovide a better result based on virus detection assay (See FIG. 1).However, when comparing the essay results between all batches and thecontrol sample (i.e., a bare virus sample), absorbance difference is notbig enough to warrant a successful coating of the virus. Thus, a changeof other condition is needed.

Example 3. Failure Attempt to Prepare Nanoparticles Comprising a VirusCore Following Known Methods with High Shear Homogenizer

Equipment and materials used are the same as in Example 2 except PBSsolution was used for the mixture of RBC membrane with the virus stocksolution. The mixture was then gone through high shear homogenizerprocess as detailed below.

Procedure:

-   -   1. Pour cold water into the cooling box of LM10 high shear        homogenizer (Microfluidics). Connect the compressed air to the        machine, adjust the air pressure to around 120 psi.    -   2. Fill the reservoir with 100 mL deionized water, set the        processing pressure to 10K psi. Wash the system with deionized        water twice and PBS twice.    -   3. Mix 14 mL PBS with 1 mL cell lipid (1.5 mg/mL) and 50 uL        adenovirus (10{circumflex over ( )}12 vp/mL, Vector Biolabs),        pour the mixture into the reservoir.    -   4. Start the machine, repeat the high shear stroke for 5 times.    -   5. Collect sample from the product container.

The experiment was not successful, no virus was detected using virusdetection assay. The virus was potentially destroyed by the high shearforce generated by the equipment. Thus, despite the successfullyapplication of RBC membrane to a core comprising non virus materials,the condition leading to a successful nanoparticle comprising a core ofan exemplary virus disclosed herein was still questionable.

Example 4: Attempts to Prepare Nanoparticles Comprising a Virus Corewith the Change of Various of Mixture Solutions

Based on the known method and scientific principles, a saline or PBSsolution of RBC membrane coating or encapsulating process is neededsince these solutions were used to prepare RBC ghosts. Contrary to theknown procedure, a few non salt solutions were used to explore the“non-traditional” procedure.

-   -   1. Add 800 uL of various solution (e.g., saline, sucrose,        dextrose, lysine-dextrose) in a 1.5 mL microcentrifuge tube.    -   2. Add 200 uL of RBC membrane (1.5 mg/mL) into the tube, mix        well. This will make process mixture with 0.3 mg/mL membrane.    -   3. Turn on the power of probe sonicator, wipe the probe with 70%        ethanol and move only the probe into the biohood.    -   4. Adjust the power of the sonicator to level 2 (output-3 W)    -   5. Prepare an ice box with cold water to keep the process        temperature at <4° C.    -   6. Add 10 uL adenovirus stock solution (Vector biolabs, Lot        #20170721.2) into the process mixture.    -   7. Put the tube into the ice box. Dip the probe into the tube.        Don't let the probe touch the bottom.    -   8. Manually sonicate the mixture with 1 second sonication and 1        second interval between each sonication. Maintain this step for        4 min to produce various of batches.    -   9. Follow vendor's procedure (Virusys Corporation, AK290-2) to        obtain the sandwich ELISA data for confirmation and comparison        of the prepared batches.

Based on the known procedure described in US 2013/033 7066, saline orPBS should be used to prepare nanoparticles. However, as clearly shownin FIG. 2, the saline batch yielded very poor results of encapsulated(or coated) nanoparticles as the ELISA assay showed almost the sameabsorbance in comparison with the uncoated virus (control). On the otherhand, unexpectedly and surprisingly, the non-salt solution batches suchas sugar containing solution batches (e.g., sucrose, dextrose, andlysine-dextrose) exhibit much lower absorbance indicating more andbetter coating of RBC membrane over the virus core.

Example 5: Coating of RBC-Membrane with an Oncolytic Virus Under anExemplary Condition

To verify the unexpectedly found results with the use of non-saltsolution for the mixture of RBC membrane with the virus stock solution,an exemplary invention procedure as shown below was used to preparenanoparticles comprising a virus core.

Equipment, materials are the same as ones in Example 2 except 11% ofsucrose solution was used for the mixture of RBC membrane with the virusstock solution.

List of Raw Materials:

Name Description Notes Adenovirus Human type 5 adenovirus From vectorbiolabs Sucrose For making 11% solution H₂O Sterile water for injectionRBC Made from RBC Membrane 1.5 mg/mL based membrane Derivation Processas in on BCA Example 1

List of Buffers and Solutions:

Name Description Quantity Sucrose solution 11% 10 mL Coating process 0.3mg/mL membrane 1 mL mixture based on BCA

Procedure:

-   -   1. Add 800 uL of 11% sucrose solution in a 1.5 mL        microcentrifuge tube.    -   2. Add 200 uL of RBC membrane (1.5 mg/mL) into the tube, mix        well. This makes process mixture with 0.3 mg/mL membrane.    -   3. Turn on the power of probe sonicator, wipe the probe with 70%        ethanol and move only the probe into the biohood.    -   4. Adjust the power of the sonicator to level 2 (output-3 W)    -   5. Prepare an ice box with cold water to keep the process        temperature at <4° C.    -   6. Add 2.5 uL exemplary oncolytic virus (e.g., adenovirus stock        solution (Vector biolabs, Lot #20170721.2) into the process        mixture, resulting about a 9% of sucrose solution of the        mixture. It is expected by a skilled person in the art that 5%        to 15% of sugar solution would work in the similar condition.    -   7. Put the tube into the ice box. Dip the probe into the tube.        Don't let the probe touch the bottom.    -   8. Manually sonicate the mixture with 1 second sonication and 1        second interval between each sonication. Maintain this step for        4 min to prepare nanoparticles comprising a core of the virus.    -   9. For the bare virus, follow steps 1-9, but change the solution        in 2. to molecular grade water.

The resulted coated nanoparticles were imaged by Transmission electronmicroscopy (TEM). FIG. 3 shows the image comparison between the barevirus and RBC membrane coated virus. The particle size comparison isshown in FIG. 4, which suggests the coating layer is about 10 nm. TheTEM image show the size of nanoparticles is in a range of 110 nm to 120nm (about 118 nm).

FIG. 5 shows that RBC membrane coated virus was protected from detectionantibody while the bare virus was clearly shown; the absorbancedifference is very significant indicating the very good RBC membranecoating result. Next the coated nanoparticles went through a process toremove the RBC membrane and then subject to the same ELISA test tofurther confirm the coating result.

For the coating removal, mix 200 uL of coated virus with 50 uL of samplepreparation buffer (Virusys Corporation, contains surfactant) beforeprocessing ELISA. FIG. 6 shows that after removal of the RBC membrane,the viruses were detected again, confirming the prior coated viruses.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A nanoparticle comprising: an inner corecomprising an oncolytic virus; and an outer surface comprising acellular membrane derived from a red blood cell, wherein internalproteins of the red blood cell have been depleted.
 2. The nanoparticleof claim 1, wherein said oncolytic virus is herpesvirus, vaccinia virus,reovirus, adenovirus, measles virus, parvovirus, or combinationsthereof.
 3. The nanoparticle of claim 1, wherein said oncolytic virus isan adenovirus.
 4. The nanoparticle of claim 1, wherein the nanoparticlehas a diameter ranging from about 110 nm to about 200 nm.
 5. A method ofmaking a nanoparticle, said method comprising: providing an oncolyticvirus; providing a red blood cell membrane, wherein internal proteins ofthe red blood cell have been depleted; mixing the oncolytic virus withthe erythrocyte membrane in a non-salt water solution; and applyingsonication to said solution of mixture to form a nanoparticle comprisingan inner core comprising said oncolytic virus and an outer surfacecomprising said erythrocyte membrane.
 6. The method of claim 5, whereinsaid non-salt water solution comprises sugar, or a component withsimilar property of a sugar in water solution.
 7. The method of claim 5,where said non-salt water solution is a sugar solution.
 8. The method ofclaim 7, wherein said sugar solution is sucrose, or dextrose containingsolution.
 9. The method of claim 7, wherein the sugar solution is asucrose solution ranging from about 5% to about 15% sucrose.
 10. Themethod of claim 5, wherein said oncolytic virus is herpesvirus, vacciniavirus, reovirus, adenovirus, measles virus, parvovirus, or combinationsthereof.
 11. The method of claim 10, wherein said oncolytic virus isadenovirus.
 12. The method of claim 5, wherein applying sonicationcomprises interval sonication for about four minutes.